THE ARTIFACT OF MANAGED HEALTH CARE

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This article highlights a common and disturbing artifact in surgical pathology. We suspect that many of you will relate to Figure 1, and hope that you will see something of use in the report on our research covering this problem, detailed within. As your lab comes under increasing pressure to produce more in less time, with fewer people, keep us in mind. Your problems are our challenges. We have a variety of solutions that will give your lab the competitive edge it needs. High quality specimens and fast turnaround time are no longer mutually exclusive goals.

Figure 1. Inadequate fixation in NBF.

Competition. Efficiency. Cost reduction. Profit. Whatever the reason, the newly evolved strategies for managed health care have put pressures on laboratories that traditionaltechnolog was never meant to accommodate. The violation of a single principle, adequate fixation, has led to a group of artifacts that was virtually unknown twenty years ago. Of the many sets of technical problems that we have been asked to solve over the years, this has come to dominate our efforts. People describe it variously as hazy or smoky, light blue, or simply washed out. It usually appears in areas rich in nuclei, especially epithelial nuclei, but it can also occur in collagen and other eosinophilic regions.

The photomicrographs in Figure 1. depict several representative examples from actual surgical pathology laboratories. While the complaint usually centers on the pale areas, the rest of the specimen is far from good. Note how most of the nuclei are merely dark blobs, lacking chromatin patterns. Others simply seem to fade away individually or in clusters.

Because histology involves so much manual manipulation of specimens, shortcuts are easy to implement. We rarely stop to consider the consequences of streamlining. You can open a tissue processor to introduce late-arriving specimens long after the program has started. You can reduce fixation to an hour or two, or omit it altogether. As long as a section can be obtained, we consider that the process has worked. But look at what we produce!

Somewhere along the way, we have forgotten what the "Old Masters" taught us many years ago. The grand figure in American histochemistry, R. D. Lillie, asserted that neutral buffered formalin (NBF) "fixes adequately in 48 hours" at room temperature, in a volume of fluid 15-20 times that of the specimen (Lillie 1965). His British counterpart, A. G. E. Pearse, wanted at least 24 hours (Pearse 1968). We laugh now at the impracticality of that in today's managed care environment, yet laughs do not obviate scientific reality. As many of the specimens that we see prove, NBF is not working.

Neutral buffered formalin does work!

As part of a research project to understand these artifacts, we fixed specimens in NBF for varying times ranging from an hour to a month. We processed them in open and closed processors set every which way. In the end, we concluded that Lillie was right. NBF needs a lot of time. A full day was not even sufficient to protect the specimen from the rigors of a closed processor. Given an appropriate volume of fixative, 40 hours of fixation was the minimum needed to produce classic morphological images on a consistent basis. (Figure 2)

Figure 2. 40 hour fixation in NBF, intestine.

With such treatment, staining is unmarred by the odd colored patches seen in Figure 1. Notice the exquisite detail in Figures 2 and 3. This is what specimens should look like should look like routinely, but they fail to do so with increasing frequency today.

There is nothing wrong with neutral buffered formalin, as these photomicrographs show. It simply is not suited to today's clinical laboratory environment. The fact that it is probably the worst common fixative for immunohistochemistry is another matter altogether.

Figure 3. 40 hour fixation,NBF, thymus.

Why are these artifacts so common today? Without lengthy fixation (24-48 hours), macromolecules remain pliable. As various denaturing forces come along, molecules are further fixed, but not in a way that is desirable. Alcohol, xylene and heat from the processor and slide drier all have the ability to change macromolecular structure. Delicate chromatin patterns become blue blobs as weakly fixed nuclear proteins are dried, twisted, shrunk and otherwise altered by the forces of specimen preparation. The degree of fixation and the strength of these other forces combine to create a bewildering array of artifacts that all stem from the same basic problem: not enough time.

Meeting The Challenge

There are several ways to meet the challenges of managed care without compromising quality. Which one is for you depends upon your own unique situation.

First and foremost, the volume of fixative must be 15-20 times that of the specimen. Adhering to this classic rule is the easiest remedy that can be made. Lower ratios result in dilution of the fixative by fluids in the tissue, thus slowing penetration and chemical reactivity. Figure 4 depicts this volume ratio as well as an unfavorable ratio. Do your specimens compare favorably?

Figure 4a.

Fixative is 20 times the volume of the specimen.

More aggressive measures may be needed

It should be obvious that a faster, more effective fixative is needed when total fixation time is less than 24 hours. Over the years, we have developed several families of products to meet the ever changing demands of modern surgical pathology. Our research and the experiences of our customers have verified that beautiful fixation can be achieved in remarkably short time.

Figure 4b.

The fixative is only 5 times the volume of the specimen.

Our top recommendation

Figure 5. Intestine, Prefer

Our all-around favorite is Prefer, a formalin-free fixative that produces striking morphological detail in very rapid time. Small biopsies can be fixed in 45 minutes (Strine et al. 1996) when using a gentle processing schedule, although we usually recommend 1-2 hours to provide a greater margin for harsher processing conditions. Most surgical specimens are nicely fixed in 4-6 hours if grossed properly. As with alcoholic formalin, alcoholic Prefer can be used to speed things up even more, especially with fatty specimens.When used as directed, Prefer will produce specimens of remarkable clarity . Because macromolecules are properly denatured, they hold up well to the hazards of subsequent specimen preparation. Interestingly, immunoreactivity is maintained at a high level, so that antigen recovery procedures are rarely needed.

Figure 6. Thymus, Prefer

Close comparison of Figures 3 and 6 reveals that chromatin patterns are more crisp with Prefer, although slightly coarser than those seen with NBF. The major difference between Prefer and formalin is the lysis of red blood cells. Aside from a loss of bright red color, this should present no problem. What you gain in better architectural and cellular preservation more than makes up for it.

		Tissues that are difficult to section with NBF often are much improved after fixation in Prefer. For example, rat esophagus is keratinized. When hardened after several days of exposure to NBF, it may be frustrating to cut a section without the superficial layers of epithelium tearing away. Human skin presents similar problems. As you can see in the figure at left (Prefer, rat), the epithelium is thick and definitely keratinized, yet remains intact. Prefer really shines when you consider its safety features. There is no inhalation risk. It has a mild, non-irritating odor. Monitoring and other OSHA-mandated issues governing the use of formalin are not applicable. Just wear gloves and protect your eyes. After all, Prefer will fix fingers and corneas as efficiently as it does surgical specimens.
Figure 7. Rat esophagus, Prefer

Another suggestion

If you would rather stay with formalin, then switching to a zinc formalin solution could improve the situation. Unfortunately, most zinc formalins have created problems in closed tissue processors because of their tendency to precipitate in the presence of alcohol. Our three families of zinc solutions will not do this.

ANATECH's Zinc Formalin is unbuffered, but despite its acidity, formalin pigment is rarely seen with short fixation times. Z-Fix is a buffered version designed to eliminate the creation of formalin pigment even with extended storage of specimens. It has an alcoholic version, available as a concentrate, that speeds processing of fatty specimens.

Figure 8. Intestine, Z-Fix

With any of these zinc products, specimens of exceptional quality are obtainable in 6-8 hours (shorter for biopsies). Cellular detail is sharper than with NBF, and you will not see nuclear bubbling artifacts (Figures 8 and 9). Immunoreactivity is preserved as with Prefer, so you can generally omit antigen recovery.

Figure 9. Lymph node, Z-Fix

Want B-5 results without the mercury?

We have several ways to get the clarity of B-5 without the health and environmental risks of mercury.

Prefer is every bit as fast as B-5 and can be incorporated into a very brief program that produces a slide in 4-6 hours from receipt of the specimen (Strine et al. 1996).

	Our unbuffered Zinc Formalin or buffered Z-Fix will more closely duplicate the colorsof B-5 (Figure 10). Minimum fixation is several hours; some labs suggest 8 hours or overnight.
Figure 10. Bone Marrow, Zinc Formalin
Z-5 comprises our third family of zinc fixatives. If you need a fast fixative to replace B-5 for bone marrow biopsies and lymph nodes, this could be your choice. Use the same fixation protocol that you currently have for B-5. Because there is no precipitate, sections will cut more easily and de-Zenkerization is a thing of the past. As with B-5, some antibodies may require antigen recovery.

	Figure 11. Bone Marrow, Z-5

Today, laboratories are in a tough competitive struggle. Fast turnaround time and quality results are two factors that could tip the competitive balance in your favor. Give us a call if you think our solutions make sense. We would love to help make you look good.